It is proposed to study the mechanisms of Zn/Cu, Mn- and Fe-containing superoxide dismutases by mixing solutions of these enzymes with superoxide ion maintained in a water miscible organic solvent such as acetonitrile. A specially constructed stopped flow spectrophotometer will be used in these experiments. The disappearance of super-oxide ion will be followed by the decrease in its absorbance at 250 nm, and the reaction will be studied over a wide range of conditions including temperatures to -30 degrees C and below. These extreme conditions favor the possibility of detecting intermediates in the enzymatic reactions. The methodology of studying enzyme reactions at sub-zero temperatures will be generally explored. Concomitant with the mechanistic studies of superoxide dismutases, the active centers of these enzymes will be probed by a wide variety of physical methods including EPR, ENDOR, ELDOR, NMR, and optical spectroscopies, as well as the techniques of magnetic susceptibility and X-ray crystallography. BIBLIOGRAPHIC REFERENCES: J. A. Fee, R. Bergamini, and R. G. Briggs, Observations on the Mechanism of the Oxygen/Dialuric Acid-Induced Hemolysis of Vitamin E-Deficient Rat Red Blood Cells and the Protective Roles of Catalase and Superoxide Dismutase, Arch. Biochem. Biophys. 169, 160-167 (1975). Robert A. Hannula and James A. Fee, A Simple Analog Integrator, Anal. Biochem. 63, 279-280 (1975).